Increased levels of hydrogen peroxide (H2O2) is thought to occur under conditions of tissue reperfusion with oxygenated media after hypoxia. The vascular endothelium is the first line of exposure to circulating hemoglobin-based blood substitutes. We have investigated the effects of a variety of modified hemoglobins on the integrity of bovine aortic endothelial cells (BAECs) and on the H2O2- mediated cytotoxicityWe investigated the toxicity of hemoglobin/myoglobin on endothelial cells under oxidative stress conditions that include cellular hypoxia and reduced antioxidant capacity. Bovine aorta endothelial cells (BAECs), grown on microcarrier beads, were subjected to cycles of hypoxia and reoxygenation in a small volume of media, and endothelial cell monolayers were depleted of their intracellular glutathione (GSH) by treatment with buthionine sulfoximine (BSO). Incubation of diaspirin cross-linked hemoglobin (DBBF-Hb) or horse skeletal myoglobin (Mb) with BAECs subjected to 3 hours of hypoxia caused transient oxidation of the hemoproteins to the ferryl form (Fe4+). Formation of the ferryl intermediate was decreased in a concentration-dependent manner by the addition of L-arginine, a substrate of NO synthase, after 3 hours of hypoxia. Optimal inhibition of ferryl formation, possibly due to the antioxidant action of NO., was achieved with 900 mM L-arginine. Addition of hydrogen peroxide (H2O2) to GSH-depleted cells in the presence of DBBF-Hb or Mb significantly decreased cell viability. Ferryl Mb, but not ferryl DBBF-Hb, was observed in samples analyzed at the end of treatment which may explain the greater toxicity observed with Mb as opposed to DBBF-Hb. This model may be utilized to identify causative agent(s) associated with hemoprotein cytotoxicity and in designing strategies to suppress or control heme-mediated injury under physiologically relevant conditions. Indeed we were able to monitor in this model system the transition in the rdox status of large number of chemically and genetically modified hemoglobins and myoglobins and were able to identify the ferryl heme in mediating cellular toxicity, under physiologically relevant oxidative stress conditions. we are currently and in colloboration with Essex university, UK and University of Henri Poincare, France working on identify these oxidation products using EPR in blood from animals infused with some modified hemoglonins.